Protein Concentration Dependence of Palmitate Binding to β-Lactoglobulin

Abstract

The binding of palmitate to β-lactoglobulin at protein concentrations ranging from 1 to 200 μM was determined using an ultrafiltration method with [14C]palmitate. Fit of the data to theoretical models required the assumption of two independent sets of binding sites; however, binding characteristics were dependent on the protein concentration. A model assuming one set of sites on the protein monomer and another on the dimer was consistent with the data. The analysis suggests that 2 mol of palmitate are bound/mol of dimer and that the binding constant is of the order of 105 M-1; a larger number of palmitate molecules are bound per mole of monomer with a smaller binding constant of the order of 104 M-1. Apparently, formation of the dimer, by hydrophobic interactions at the monomer contact site, eliminated palmitate binding sites on the monomer but formed a higher affinity pocket for binding to the dimer.